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Volume 4, Number 2—June 1998

Letter

Detection of Glycoprotein of Burkholderia pseudomallei

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To the Editor: Melioidosis, a potentially fatal disease that is difficult to diagnose and treat, is common in areas with subtropical climate (e.g., Singapore, the southern provinces of China) and is hyperendemic in Thailand. The etiologic agent, Burkholderia pseudomallei (Pseudomonas pseudomallei), is widely distributed in Southeast Asia and northern Australia. The agent has the potential to become established in regions with similar climate conditions, particularly if animals infected with B. pseudomallei are imported from endemic-disease zones (1-3).

Rapid and reliable detection of B. pseudomallei and its antigens has many potential applications. Recently, we developed a monoclonal antibody immunoenzyme test system for the detection of minimal concentrations of a B. pseudomallei glycoprotein, which is considered one of the pathogenicity factors for this microorganism. This glycoprotein, called Ag8 by N.N. Piven and V.I. Ilyukhin (4), is present in different strains of B. pseudomallei and B. mallei but not in other Burkholderia spp. (B. aeruginosa, B. putida, B. cepacia, B. malthophilia, B. fluorescens, B. pseudoalcaligenes). Ag8 is composed of 10% protein and 90% carbohydrate, has molecular mass 800 kDa, and is localized in an extracellular capsulelike substance surrounding B. pseudomallei cells (5).

We developed an immunoenzyme test system with three monoclonal antibodies (Mab) to different epitopes of Ag8 (Mab 2A6-IgG3, Mab 2H7-IgG1, Mab 1G2-IgG2b) and one antibody to epitopes common for Ag8 and LPS of B. pseudomallei (mab 1ES-IgG2b). A sandwich enzyme-linked immunosorbent assay (ELISA) was used for the detection of Ag8 in different test samples (6). The sensitivity of the immunoenzyme test system was determined with a standard antigen sample. Minimal sensitivity (37 ng/ml of carbohydrate) was observed when polyclonal immunoglobulins were used as "catching" antibodies. Maximal sensitivity (0.37 ng/ml of carbohydrate) was noted when either Mabs 2A6 or mixtures of Mabs were used as catching antibodies.

The test system was further evaluated with samples of extracellular antigens (extracts of cultural media, fractions after gel chromatography of extracellular antigens) and bacterial suspensions of B. pseudomallei and B. mallei strains isolated in different regions of the world. Levels of Ag8 in cultural media varied considerably depending on periods of cultivation of bacteria. Additionally, the level of Ag8 varied among strains of B. pseudomallei and B. mallei. Among 61 strains of B. pseudomallei from the museum collection (most of which were isolated in Southeast Asia and northern Australia), three had increased ability to produce Ag8. These strains had been isolated from clinical specimens (blood, abscesses of hospitalized melioidosis patients) in Vietnam. The strains gave results typical of B. pseudomallei species in all routine serologic tests (agglutination test, immunofluorescence assay, immunodiffusion test). In contrast, the B. pseudomallei glanders agent (16 strains from the museum collection) had reduced ability for Ag8 production; ELISA titers of Ag8 were a thousandfold less in culture fluids in these strains.

The ELISA technique not only facilitates diagnosis of disease but also provides a rational basis for selecting strains for vaccine production. It also has considerable utility for studying the pathogenicity of B. pseudomallei.

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